CZ224295A3 - Peptide, pharmaceutical and cosmetic preparations containing thereof as well as process for preparing such peptide - Google Patents

Abstract

Disclosed herein are peptide derivatives of the formula A-B-D-NHCH{CH2C(O)R<1>}C(O)-NHCH{CR<2>(R<3>)-COOH}C(O)-E wherein A is a lower alkanoyl bearing two substituents, each substituent selected independently from phenyl or a monosubstituted phenyl wherein the monosubstituent is alkyl, halo, hydroxy or alkoxy; B is a N-methyl amino acid residue; D is an amino acid residue; R<1> is alkyl, cycloalkyl, a monosubstituted amino or a disubstituted amino; R<2> is hydrogen or alkyl and R<3> is alkyl, or R<2> and R<3> are joined to form a cycloalkyl; and E is a terminal unit, for example, an alkylamino or a monovalent amino acid radical such as NHCH(alkyl)C(O)OH. The derivatives are useful for treating herpes infections.

Description

The peptide, pharmaceutical composition and cosmetic composition of the peptide and method for producing the peptide.

Technical field

The invention relates to peptide derivatives with antiviral effects and to compositions for use in the treatment of viral diseases. More specifically, the invention relates to peptide derivatives (herein referred to as peptides) that are active against herpes viruses (herpes viruses), pharmaceutical compositions containing the peptides, and methods of using the peptides to inhibit herpes virus replication and to treat herpes infections.

BACKGROUND OF THE INVENTION

Herpes viruses cause a wide range of human and animal diseases. For example, Herpes simplex viruses, types 1 and 2, (HSV-1 and HSV-2) are responsible for cold sores and genital lesions; Varicella zoster viruses (VZV) cause chicken plague and shingles; and Epstein-Barr virus (EBV) induces infectious mononucleosis.

Over the last two decades, researchers have received the greatest attention of purine and pyrimidine nucleoside analogues in the search for agents for the treatment of herpes infections. As a result, several nucleoside analogs have been developed into antiviral agents. At the same time, acyclovir, which is the recommended agent for the treatment of genital infections caused by Herpes simplex virus, is the most successful.

However, despite some significant advantages, there remains a need for effective, safe therapeutic agents to treat herpes viral infections. An overview of current therapeutic agents in this field is provided by M.C. Naked in Antiviraí drugs; Pharmacokinetics, Adverse Effects and Therapeutic Use, J. Pharm. Technol. 3, 100, 1987.

This application discloses a group of peptide derivatives having activity against herpes viruses. The selective effect of these peptides on herpes viruses, coupled with a wide margin of safety, makes these peptides as desirable substances to combat herpes infections.

The following references disclose peptides or peptide derivatives associated with antiherpetic activity:

B.M. Dútia et al., Nature 321,439 1986, ......

E.A. Cohen et al., Nature 321, 441, 1986,

J.H. Shubak-Sharpe et al., UK patent application 2185024, published Jul.

Ί987 ------— ........... -....................

P. Gaudreau et al., J. Biol. Chem. 262, 12413, 1987.

E.A. Cohen et al., U.S. Patent 4,795,740, 3.1. 1989,

R. Freidinger et al., U.S. Patent 4,814,432, Mar. 21, 1989,

Garskey, V. M., U.S. Pat. No. 4,837,304, Jun. 6, 1989,

R. Colonno et al., U.S. Patent 4,845,195, Jul 4, 1989,

P. Gaudreau et al., J. Med. Chem. 33, 723 (1990),

J, Adams et al., European Patent Application 408,973, published Jan. 23, 1991, P.L. Beaulieu et al., European Patent Application 411,332, published Feb. 6, 1991,

J. Adams et al., European Patent Application 411,333, published February 6, 1991,

J. Adams et al., European Patent Application 411,334, published February 6, 1991, R.L. Tolman et al., European Patent Application 412,595, published February 13, 1991,

W. T. Ashton et al., European Patent Application 4338,873, published Jul. 31, 1991,

P.L. Beaulieu et al., European Patent Application 461,546, published Dec. 18, 1991,

P. Gaudreau et al., J. Med. Chem. 35, 346.1992,

-3R. Deziel and Y: Guindon, Canadian Patent Application 2,033,488, published July 1, 1992,

L.L. Chang et al., Bioorganic & Medicinal Chemistry Letters 2, 1207, 1992.

The peptides of the foregoing works can be distinguished from the peptides of the present application on the basis of characteristic structural and biological differences.

Abbreviations and symbols used in this application are set forth in the Summary of the Invention.

SUMMARY OF THE INVENTION

The peptides of the invention are represented by Formula 1

ABD-NHCH {CH ₂ C (O) R% (O) -NHCH {CR ² (R ³ ) C (O) OH} C (O) -E CD where

A is a double-substituted lower alkanoyl wherein each substituent is independently selected from the group consisting of phenyl and a single-substituted phenyl whose substituent is selected from the group consisting of lower alkyl, halo radical, hydroxy; and lower alkoxy;

B is N (CH ₃ ) CHR ⁴ C (O) wherein R ⁴ is lower alkyl; D is NH-CHR ^S C (O) wherein R ⁵ is lower alkyl or lower alkyl substituted by one carboxyl, hydroxyl, mercapto or benzyloxy group;

R ¹ is lower alkyl, lower cycloalkyl, {1 - (lower alkyl) - (lower cycloalkyl)}, or NR ⁶ R ^7, wherein ^R is hydrogen or lower alkyl and R ⁷ is lower alkyl; or R ⁶ and R ⁷ together with the nitrogen atom to which they are attached form a pyrrolidine, piperidine, morpholine or 4-methylpiperazine group;

R ² is hydrogen or lower alkyl and R ³ is lower alkyl, or R ² and R ³ together with the carbon atom to which they are attached form a lower cycloalkyl; and E is NHR® wherein R ⁸ is C _{4th 9} alkyl; lower cycloalkyl; lower cycloalkyl, monosubstituted or

-4-lower alkyl or substituent (lower alkyl) - (lower cycloalkyl); or E is NHCHtR ^-Z, where R ⁹ is C ₄ . _with alkyl, lower cycloalkyl or (lower cycloalkyl) - (lower alkyl) and Z is CH ₂ OH, C (O) OH, C (O) NH ₂ or C (O) OR ¹⁰ , wherein R ¹⁰ is lower alkyl ; or a therapeutically acceptable salt thereof.

Ϊ

A preferred group of peptides of the invention is represented by Formula 1 wherein A is lower alkanoyl, double substituted with phenyl, 4- (lower alkyl, phenyl, 4-haloenyl, or 4- (lower alkoxy) phenyl; B is (N-Me) Val, (N-Me) li or (N-Me) Tbg; D is the amino acid residue of (S) -2-amino-3-hydroxy-3-methylbutyric acid or (R) -2-amino-3-mercapto- A 3-methylbutyric or amino acid residue selected from Val, Ile and Tbg; R 6 is lower-lower cycloalkyl, {1- (loweralkyl) lower alkyl}, Ν, ím-dimethylamino, Ν, Ν-diethylamino group, pyrrolidino or morpholino group; R ² and R ³ are as defined above; ^& aEje NHR, wherein R ⁸ is C4.3 alkyl, lower cycloalkyl, lower cycloalkyl monosubstituted or disubstituted with lower alkyl / substituent or (lower alkyl or E is NHCH (R ^S ) -Z, wherein R ⁹ is C 1-4 alkyl or (lower cycloalk); yl) methyl and Z are as previously defined, or a therapeutically acceptable salt thereof.

A more preferred group of peptides is represented by Formula 1, wherein A is

2- (phenylmethyl) -3-phenylpropionyl, 2 - {(4-fluorophenyl) methyl} -3- (4-fluorophenyl) propionyl, 2 - {(4-methoxyphenyl) methyl} -3-phenylpropionyl or 2- {(4-methoxyphenyl) methyl} -3- (4-methoxyphenyl) propionyl; B is (N-Me) -Val or (N-Me) -lle; D is Val, Lie or Tbg; R ¹ is 1-methylethyl, 1,1-dimethylethyl, 1,1-dimethylpropyl, cyclobutyl, cyclopentyl, cyclohexyl, 1-methylcyclopenthyl, N, N-dimethylamino, Ν, Ν-diethylamino, pyrrolidine or morpholine; R ² is hydrogen and R ³ is methyl, ethyl, 1-methylethyl, 1,1-dimethylethyl or propyl, and the carbon atom bearing R ² and R ³ is in the (R) configuration, or R ² and R ^{3 are} each independently methyl or ethyl, or R ² and R ³ together with the carbon atom to which they are attached form a cyclobutyl, cyclopentyl or cyclohexyl; and E is NHR ⁸ wherein R ⁸ is 2,2-dimethylpropyl,

-51 (R), 2,2-trimethylpropyl 1,1 (R) -ethyl-2,2-dimethylpropyl, 2,2-dimethylbutyl,

3,3-dimethylbutyl, 1 (R), 2,2-trimethylbutyl, 1 (R), 3,3-trimethylbutyl,

1 (R) -ethyl-3,3-dimethy [, or cyclohexylmethyl; or E is NHCH 2 R 2 -Z, wherein the carbon atom bearing R ⁹ is in the (S) configuration, R ⁹ is 1,1-dimethylethyl,

1-methylpropyl, 2-methylpropyl, 2,2-dimethylpropyl or cyclohexylmethyl and 2 is CH ₂ OH, C (O) OH, C (O) NH ₂ or C (O) OR ¹⁰ , wherein R ^w is methyl, ethyl, or propyl; or a therapeutically acceptable salt thereof.

The most preferred group of peptides is represented by Formula 1, wherein A is

2- (phenylmethyl) -3-phenylpropionyl; B is (N-Me) Val; D is Tbg; R ¹ is 1-methylethyl,

1,1-dimethylethyl, 1-methylpropyl, 1,1-dimethylpropyl, 2,2-dimethylpropyl, cyclobutyl, cyclopentyl, cyclohexyl, 1-methylcyclopentyl, pyrrolidine or morpholino; R ² is hydrogen and R ³ is methyl, ethyl, 1-methylethyl or propyl, and the carbon atom bearing R ² and R ³ is in the (R) configuration, or R ² and R ³ are each independently methyl or ethyl, or R ² and R ³ together with the carbon atom to which they are attached form cyclobutyl, cyclopentyl or cyclopentyl; cyclohexyl; and E is NHR ⁸ , where R ® is

2,2-dimethylpropyl, 1 (R), 2,2-trimethylpropyl, 1 (R) -ethyl-2,2-dimethylpropyl,

2,2-dimethylbutyl or 1 (R) -ethyl-3,3-dimethylbutyl, or E is ^ NHCHfR -Z wherein the carbon atom bearing R @ ⁹ is (S) -configuration, ^R is 2,2-dimethylpropyl and Z is CH ₂ OH, C (O) OH, C (O) NH ₂ or C (O) OR ¹⁰ , wherein R ¹⁰ is methyl, ethyl, or propyl; or a therapeutically acceptable salt thereof.

F

Included within the scope of this invention is a pharmaceutical composition comprising an antiviral effective amount of a peptide of Formula 1, or a therapeutically acceptable salt thereof, with respect to Herpes virus, and a pharmaceutically or veterinarily acceptable carrier.

Also included within the scope of this invention is a cosmetic composition comprising a peptide of formula 1, or a pharmaceutically acceptable salt thereof, and a physiologically acceptable carrier suitable for topical application.

An important aspect of the invention is a method of treating herpes viral infections

In mammals, which is administered to a mammal with respect to the Herpes virus an effective amount of a peptide of Formula 1, or a therapeutically acceptable salt thereof.

Another important aspect includes a method of inhibiting herpes virus replication, wherein the virus is contacted with an amount of a peptide of Formula 1, or a therapeutically acceptable salt thereof, that inhibits herpesvirus ronucleotide reductase.

Yet another aspect encompasses a method of treating herpes viral infections in a mammal comprising administering to the Herpes virus an anti-virally effective amount of a combination of a peptide of Formula 1, or a therapeutically acceptable salt thereof, and an anti-viral nucleoside analog. The present invention also includes a pharmaceutical composition comprising the combination.

Methods for making the peptides of Formula 1 are described as follows.

Formula 1 may otherwise be represented as:

The term residue in relation to an amino acid or amino acid derivative refers to a radical derived from the corresponding α-amino acid by eliminating the hydroxyl of the carboxyl group and one hydrogen atom of the α-amino group.

Abbreviations used here. .k ... labeling of amino acids and protecting groups, ·

-7 generally comply with the IUPAC-IUB Commission on Biochemical Nomenclature (IUPAC-IUB) recommendations, see European Journal of Biochemistry 138, 9, 1984. For example, Val, Ile, Asp and Leu represent residues of L-valine, L-isoleucine , L-aspartic acid and L-leucine.

Ť

AND.

* Asymmetric carbon atoms located in major straight axes (ie in the backbone, backbone) of peptides of formula 1, not including the A and Z (E) end groups, but including the carbon atom bearing R ⁹ when E is NHCHfR? -Z, as previously mentioned, have an S configuration.

Asymmetric carbon atoms, located in the side chain of an amino acid or derived amino acid residue, in terminal group A, and in terminal group E, if E represents NHR® as previously defined, may have a S or R configuration.

The designation Tbg represents the amino acid residue of (S) -2-amino-3,3-dimethylbutanoic acid. The designation yMeLeu represents the amino acid residue of (S) -2-amino-4,4-dimethylpentanoic acid. The term yMeLeucinol represents (S) -2-amino-4,4-dimethylpentanol with one hydrogen atom removed from the α-amino group.

Other designations used herein are: (N-Me) Val for the acid residue

(S) -3-methyl-2- (methylamino) butanoic; (N-Me) lle for residue acid (R) -3-methyl-2- (methylamino) -pentanoic; (N-Me) Tbg for residue acid (S) -2- (methylamino) -3,3-dimethylbutanoic; Asp (cyBu) for residue acid (S) -α-amino-1-carboxycyclobutanocyte; Asp (cyPn) for residue acid

(S) -α-amino-1-carboxycyclopentaneacetic and Asp {(R) -Me} for the 3- (R) -methyl-L-aspartic acid residue (ie {S- (R *, S *)} - 2 amino-3-methylbutanedioic acid).

The term halo, as used herein, means a halo radical selected from a bromine, chlorine, fluorine or sodium radical.

The term lower alkanoyl as used herein refers to a straight chain 1-oxoalkyl of two to six carbon atoms or a branched chain 1-oxoalkyl of two to six carbon atoms; for example acetyl, propionyl (1-oxo-propyl) and 2-methyl-1-oxopropyl.

The term C _4-6 alkyl, as used herein, refers to straight or branched chain alkyl radicals containing 4 to 9 carbon atoms, and includes, for example,

1-methylpropyl, 2-methylpropyl, 2,2-dimethylpropyl, 1,2,2-trimethylpropyl,

3,3-dimethylbutyl, 1-ethyl-2,2-dimethylbutyl and 4,4-dimethylpentyl.

The term lower alkyl as used herein, alone or in combination with another radical, refers to straight chain alkyl radicals containing 1 to 6 carbon atoms and branched chain alkyl radicals containing 3 to 6 carbon atoms and includes methyl, ethyl, propyl, butyl, hexyl, 1-methylethyl,

1-methylpropyl, 2-methylpropyl and 1,1-dimethylethyl.

The term {1- (lower alkyl) - (lower cycloalkyl)}, as used herein, refers to a lower cycloalkyl radical bearing a lower alkyl substituent at the 1-position; for example

1-ethylcyclopropyl, 1-propylcyclopentyl and 1-propylcyclohexyl,

The term lower cycloalkyl as used herein, either alone or in combination with another radical, either alone or in combination with another radical, refers to saturated cyclic hydrocarbon radicals containing three to six carbon atoms and includes cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.

The term lower alkoxy, as used herein, refers to straight chain alkoxy radicals containing one to four carbon atoms and branched chain alkoxy radicals containing three to four carbon atoms, and includes methoxy, ethoxy, propoxy, 1-methylethoxy, butoxy and the like. , 1-dimethylethoxyl radical. The latter radical is commonly known as tertiary butoxyl.

The term pharmaceutically acceptable carrier or veterinarily acceptable carrier as used herein refers to a nontoxic, generally inert binder (vehicle) of an active ingredient that does not retroactively affect the ingredient.

The term physiologically acceptable carrier, as used herein, refers to an acceptable cosmetic binder (vehicle) of one or more non-toxic excipients that do not reduce or react with the efficacy of the active ingredient contained in the binder.

The term "veterinarily acceptable carrier" as used herein refers to a physiologically acceptable binder (vehicle) for administering therapeutic agents to a pet that comprises one or more non-toxic excipients that do not reduce or react with the efficacy of the active ingredient contained in the binder.

The term effective amount refers to a predetermined antiviral amount of the antiviral agent, ie, the amount of the agent sufficient to render the agent effective against viral organisms in vitro.

The term coupling agent as used herein refers to an agent capable of effecting binding of the free carboxyl group of an amino acid or peptide to the free amino group of another amino acid or peptide, releasing water and forming an amide bond between the reactants. Similarly, such an agent can effect an acid-alcohol bond to form the corresponding esters. The agents allow or facilitate dehydration binding by activation of the carboxyl group. Descriptions of such coupling agents and activated groups are contained in general peptide chemistry textbooks; for example, in E. Schroder and K.L. Lubke The Peptides, Volume 1, Academic Press, New York, NY, 1965, pp. 2-128, and in KD Kopple's Peptides and Amino Acids, edited by WA Benjamin, Inc., New York, NY, 1966, p.33-51. Examples of coupling agents include diphenylphosphoryl azide, Ι, Γ-carbonyldiimidazole, dicyclohexylcarbodiimide, N-hydroxysuccinimide or 1-hydroxybenzotriazole in the presence of dicyclohexylcarbodiimide. A very practical and useful coupling agent is hexafluorophosphate (benzotriazol-1-yloxy) -tris-10 (dimethylamino) phosphonium, described by B. Castro et al. In Tetrahedron Letters 1219, 1975, see also D. Hudson in J. Org. Chem. 53, 617, 1988, either alone or in the presence of 1-hydroxybenzotriazole. Yet another very practical and useful binding agent is commercially available tetrafluoroborate

2- (1H-benzotriazol-1-yl) -N, N, N ', N'-tetramethylurea.

Method

The peptides of Formula 1 may be prepared by methods that include methods generally used in peptide synthesis, such as classical coupling of amino acid residues and / or peptide fragments in solution. Such methods are described, for example, by E. Schroeder and K. Lubble, supra, in the series of The

Peptides: Analysis, Synthesis, Biology, edited by E. Gross et al., Academic Press, New York, Ύ.Ύ., 1979-1987, parts 1 to 8, and J.M. Stewart and J.D.Young in Solid Phase Peptide Synthesis, 2nd Edition, Perition Chem. Co., Rockford, IL, USA,

1984.

A general feature of the aforementioned peptide procedures is the protection of the reactive groups of various amino acid residues or derived amino acid residues (or, if desired, non-peptide fragments of the peptide) in the side chain with suitable protecting groups that will prevent chemical formation at that end. reaction until final removal. protecting groups. It is also common to protect the .alpha.-amino group of an amino acid or fragment for as long as the particle reacts via its carboxyl group and subsequently selectively remove the .alpha.-amino protecting group so that a subsequent reaction can take place at this point. Another common feature is the initial protection of the C-terminal carboxyl of an amino acid residue or peptide fragment, if present, to become the C-terminal functional group of the peptide, with a suitable protecting group to prevent chemical reaction at this point until deprotection. after assembly.required p.eptide sequences .. .. ..........

Thus, in general, the peptide of Formula 1 can be prepared by linking in a step-by-step sequence, according to the sequence of peptides, appropriate amino acids or derived amino acid residues and non-peptide fragments of peptides (such as key intermediates), which are suitably protected if necessary and removed All protecting groups, if present, upon completion of the multi-step binding to obtain the peptides of Formula 1. More specific procedures are illustrated in the Examples below.

Most of the intermediates and processes for preparing some of these have been described by J. Adams and co-authors in European Patent Application No. 411 332, published February 6, 1991, by J. Adams and co-authors in European Patent Application No. 411 334, published on February 6, 1991 and R, Deziel and Y. Guindon in Canadian Patent Application No. 2,033,448, published July 1, 1992.

The lower aikanoic acids, disubstituted by phenyl or monosubstituted by phenyl, required to form the N-terminus of the present peptides are known or can be prepared by known methods; for example, see LL. Chang et al., Bioorganic and Medicinal Chemistry Letters 2, 1207, 1992. Dibenzylacetic acid was described by M.R. Doliquem in Ann. Chim. (Paris) 15 (10), 425 (1931) and F. Krolipfeiffer and A. Rosenberg, Chem. Ber. 69,465,1936.

The peptide of formula 1 of the invention may be obtained in the form of a therapeutically acceptable salt. Where a particular peptide has a base-acting residue, examples of such base salts with an organic acid such as acetic, lactic, succinic, methanesulfonic or p-toluenesulfonic acid, as well as polymeric acids such as tannic acid or carboxymethylcellulose, as well as salts with inorganic acids such as hydrohalic acids, for example hydrochloric acid, or sulfuric or phosphoric acid. If desired, a particular acid addition salt is converted to another acid addition salt, such as a non-toxic, pharmaceutically acceptable salt, by affecting the respective ion exchange resin as described by RA Boissonnas and co-authors v

-12Helv. Chim. Acta 43, 1849 (1960).

Where a particular peptide has one or more free carboxyl groups, examples of such salts are carboxylic acid salts with sodium, potassium or calcium cations, or with organic bases such as triethylamine or N-methylmorpholine.

Anti-herpes activity

The antiviral activity of the peptides of Formula 1 can be demonstrated by biochemical, microbiological and biological procedures; showing the inhibitory effect of compounds on the replication of Herpes simplex viruses (plain herpes) type 1 and 2 (HSV-1 and HSV-2) and on other Herpes viruses, such as Varicella zoster (VZV) and Epstein-Barr viruses (EBV) ).

In the examples below, the inhibitory effect of herpetic ribonucleotide reductase is exemplified by the peptides of formula 1. In connection with said specific inhibition of herpetic ribonucleotide reductase, it is noteworthy that the peptides have relatively little or no effect on cellular ribonucleotide reductase activity required for normal cell replication.

A method for demonstrating the inhibitory effect of the peptides of Formula 1 on viral replication is the cell culture technique; see, for example, Canadian Patent Application No. 2,033,488 to R. Deziel and Y. Guindon, published July 1, 1992.

The therapeutic effect of the peptides can be demonstrated in laboratory animals, for example using the herpes simplex virus-induced eye disease assay in mice, described by C.R. Brandt and co-authors in J. Virol. Meth. 36, 209 (1992).

When the peptide of the invention is one of its therapeutically acceptable

The salts, used as an antiviral agent, are administered topically or systemically to warm-blooded animals, such as humans, pigs or horses, in a vehicle containing one or more pharmaceutically acceptable carriers whose proportion is determined by the solubility and chemical nature of the peptide selected by administration and standard biological practice. . For topical administration, the peptide may be formulated in pharmaceutically acceptable vehicles containing 0.1 to 5% and preferably 0.5 to 2% active agent. Such formulations may be in the form of a solution, cream or emulsion.

For systemic administration, the peptide of Formula 1 is administered by intravenous (vein), subcutaneous (subcutaneous) or intramuscular (intramuscular) injection, in compositions with pharmaceutically acceptable vehicles or carriers. For injection, it is preferred to use the peptide in solution in a sterile aqueous vehicle, which may also contain other solutes, such as buffers or preservatives, as well as sufficient amounts of pharmaceutically acceptable salts or glucose to render the solution isotonic.

Suitable vehicles or carriers for the above compositions are described in standard pharmaceutical texts, for example, in Remington's Pharmaceutical Sciences, 18th Edition, Mack Publishing Company, Easton, Penn., 1990.

and

The dosage of the peptide will vary with the form of administration and the particular active agent selected. In addition, it will vary with the particular host being treated. Generally, treatment is initiated in small increments until the optimum effect is achieved under the given conditions. Preferably, the peptide is administered at a concentration that generally provides antiviral activity without causing any harmful or deleterious side effects.

For topical administration, the peptide is applied to the skin in a suitable, topically administered formulation on an infected part of the body, e.g., on the skin or part of the body.

-14 the oral or genital cavity, in sufficient quantity to cover the infected area. Treatment should be repeated, eg every 4 or 6 hours, until the lesion has healed.

For systemic administration, the peptide of Formula 1 is administered at a dose of 10 µg to 500 µg per kilogram of body weight per day, although the above variations may also occur. However, to achieve effective results, it is preferable to use a dosage of about 10 to 200 pg per kilogram of body weight per day.

Another aspect of the invention includes cosmetic compositions comprising an amount of the peptide of Formula 1, or a therapeutically effective salt thereof, that acts prophylactically against the Herpes virus, together with a physiologically acceptable cosmetic carrier. Additional ingredients such as skin emollients may also be included in the formulation. The cosmetic composition of the present invention is used prophylactically to prevent the appearance of herpes lying on the skin. The composition can be applied overnight to the appropriate areas of the skin. Generally, the cosmetic composition contains less peptide than the corresponding topical pharmaceutical compositions. The preferred amount of peptide in the cosmetic composition is in the range of 0.01 to 0.2 weight percent.

Although the above described compositions are said to be effective and relatively safe drugs for the treatment of herpes viral infections, the possibility of co-administering these compositions with other anti-viral drugs or agents for better results is not excluded. Such other anti-viral drugs or agents include anti-viral nucleosides such as acyclovir , and antiviral surfactants or antiviral interferons such as those described by SS Asculai and F. Rapp in U.S. Patent No. 4,507,281.26.3. 1985.

More specifically, with respect to the treatment of herpes viral infections by concomitant administration, it has been found that the antiherpetic activity of antiviral nucleoside analogues can be synergistically enhanced without concomitant increase in toxic effects by combining them with the peptide of Formula 1. Accordingly, it is presented herein

A pharmaceutical composition for treating herpes infections in mammals, comprising a pharmaceutically or veterinarily acceptable carrier and an effective amount of a combination of an antiviral nucloside analogue or a therapeutically acceptable salt thereof with a ribonucleotide reductase inhibiting peptide or a therapeutically acceptable salt thereof.

Also disclosed is a method of treating herpes viral infections in mammals. The method comprises administering to a mammal a combination of a compound of Formula 1, or a therapeutically acceptable salt thereof, and an antiviral nucloside analogue or therapeutically acceptable salt thereof, in an amount effective against the Herpes virus.

The antiviral nucleoside analogue used in combination is one that can be enzymatically converted (in vtvo) to a viral DNA polymerase inhibitor, and / or to an alternative substrate of a herpetic DNA polymerase. The antiviral nucleoside analogue may be selected from known nucleoside analogues. Preferred nucleoside analogs of the invention include acyclovir and analogs thereof; for example compounds of formula 2

CH ₂ OCH ₂ CH ₂ OH wherein R ¹¹ is hydrogen, hydroxy or amino, or a therapeutically acceptable salt of such compound. (Formula 2 wherein R ¹¹ is hydroxy is acyclovir).

Other preferred antiviral nucleoside analogues for

The uses of the present invention include vidarabine, idoxuridine, trifluridine, ganciclovir, edoxudine, brovavir, facltabine, penciclovir, famciclovir and rociclovir.

The term synergistic effect, used in relation to the antiviral or anti-herpes activity of the above defined combinations of the nucleoside analogue and peptides of formula 1, denotes an antiviral or anti-herpes activity that is higher than the anticipated sum of the effects of the two individual components of the combination.

When the combination of the invention is used to treat herpes infections, the combination is administered to a warm-blooded animal, eg, a human, swine or horse, in a vehicle containing one or more pharmaceutically acceptable carriers, the ratio of which is determined by the solubility of the of Formula 1, the selected route of administration, standard biological practice, and the proportions of two active ingredients to provide a synergistic antiviral effect. The combination is preferably applied topically. For example, the two active agents (i.e., the antiviral nucloside analogue and the peptide of Formula 1, or therapeutically acceptable salts thereof) may be formulated as solutions, emulsions, creams, or lotions in pharmaceutically acceptable vehicles. Such compositions may contain from 0.01 to 1.0 weight percent of the nucleoside analog, or therapeutically acceptable salt thereof, and about 0.05 to 1 weight percent of the peptides of Formula 1, or therapeutically acceptable salts thereof.

In any case, the two active agents are present in the pharmaceutical composition in amounts sufficient to provide a synergistic antiherpetic effect.

The following examples further illustrate the invention. Temperatures are given in degrees Celsius. The percentages or ratios of the solutions represent the volume to volume relationship unless otherwise indicated. Nuclear magnetic resonance spectra were obtained using a Bruker 200 or 400 MHz spectrometer (400 MHz mentioned in the introduction); chemical shifts (δ) are given in parts per million (ppm). Abbreviations used in the Examples include Boc: tert-butyloxycarbonyl; Bzl: benzyl; EtOH: ethanol; EtOAc:.,

-17 ethyl acetate; Et, O: diethyl ether; HPLC; high performance liquid chromatography; MeOH: methanol; THF: tetrahydrofuran.

and

DETAILED DESCRIPTION OF THE INVENTION

Example 1

General Procedure for Coupling Reactions {See also R. Knorr et al., Tetrahedron Letters 30,1927,1989}

The first reactant, i.e. the free amine (or its hydrochloride salt), is dissolved in dichloromethane or acetonitrile and the solution is cooled to 4 ° C. Under an atmosphere of nitrogen, 4 equivalents of N-methylmorpholine are added to the stirred solution. After 20 minutes, one equivalent of the other reactant, i.e. free carboxylic acid, and 1.05 equivalent of coupling reagent are added. (Practical and effective coupling agents for this purpose are hexafluorophosphate (benzotriazol-1-yloxy) tris- (dimethylamino) phosphonium, or better tetrafluoroborate

2- (1H-benzotriazol-1-yl) -N, N, N ', N'-tetramethylurea). The reaction is monitored by TLC (thin layer chromatography). Upon completion of the reaction, the dichloromethane (or acetonitrile) was evaporated under reduced pressure. The residue was then dissolved in EtOAc. The solution was then washed with 1N aqueous citric acid, 10% aqueous Na ₂ CO _3, and brine. The organic phase was dried (MgSO 4, filtered and concentrated to dryness under reduced pressure. The residue was purified on silica gel (SiO ₂ ) according to Stills flash chromatography (WC Still et al., J. Org. Chem. 43, 2923, 1978).

Example 2

Λ · ϋ

·>

Preparation of intermediate H-Asp (cyPn) (Bzl) -NH- (S) -CH {CH ₂ C (CH 3) ₃ } CH ₂ OBzl (a) (S) -α-Azido-1 - {(phenylmethoxy) carbonyl acid } -cykiopentanactová:

This compound was prepared from the 2-oxospiro [4.4] nonane-1,3-dione described by M.N. About-Enein and spoi. in Pharm. Acta Helu. 55, 50, 1980, according to the asymmetric azidation method utilizing Evans's excipients, see D.A. Evans et al., J. Amer. Chem. Soc. 112, 4011 (1990).

More specifically, under a argon atmosphere, a 1.6 M hexane solution of butyl lithium (469 mL, 750 mmol) was added dropwise to a solution of the chiral excipient, 4 (S) - (1-methylethyl) -2-oxazolidinone (96.8 g, 750 mmol). described by LN Pridgen and J. Prol in J. Org. Chem. 54, 3231, 1989) in dry THF at -40 ° C. The mixture was stirred at -40 ° C for 30 minutes and then cooled to -78 ° C. 2-Oxospiro [4,4] nonane-1,3-dione was added dropwise to the cold mixture. The mixture was then stirred for 1 hour at 0-G-a-subsequently-to-20% (w / v) aqueous citric acid solution (600 ml). The organic phase was separated and the aqueous phase was extracted with EtOAc. The combined organic phases were washed with brine, dried (MgSO 4 and concentrated under reduced pressure to give 3- (2- (1-carboxycyclopentyl) -1-oxoethyl)} - 4- (S) - (1-methylethyl) -2-oxazolidinone as a pink solid (300 g).

This solid (ca 750 mmol) was dissolved in acetonitrile (11). Benzyl bromide (128.3 g, 89.2 mL, 750 mmol) and 1,8-diazabicyclo [5.4.0] undec-7-ene (114 g, 112 mL, 750 mmol) were added to the solution. The mixture was stirred for 16 hours under an argon atmosphere. The volatiles were removed under reduced pressure and the residue was dissolved in H ₂ O / EtOAc. The organic phase was then separated, washed with 10% (w / v) aqueous citric acid solution and brine, dried (MgSO ₄ ) and concentrated to dryness under reduced pressure to give an oil. Crystallization of the oil from hexane / EtOAc gave the corresponding benzyl ester as a white solid (204 g, 73%).

A solution of the material so obtained (70 g, 137 mmol) in dry THF (200 mL) was cooled to -78 ^at _c; The cold solution should! A 0.66 M THF solution of 1,1,1,3,3,3-hexamethyldisiiazane (286 mL, 189 mmol) containing 6% (w / v) cumene was added over 15 minutes. The mixture was stirred at -78 ° C for 45 minutes. Ke cold

A solution of 2,4,6-triisopropylbenzenesulfonyl azide (67 g, 216 mmol) in dry THF (100 mL) was added in one portion, followed by glacial acetic acid (50 mL, 860 mmol) in two portions. The mixture was heated and stirred at 35-45 ° C for 1 hour. The volatiles were removed under reduced pressure. The yellow residue was triturated in hexane / EtOH (4: 1, 1.7 L). The resulting white solid was collected on a filter. The filtrate was mixed with SiO ₂ (230-240 mesh). The volatiles were removed under reduced pressure and the residual solid was dried at 35 ° C under reduced pressure to remove cumene. The remaining solid was then placed on a SiO ₂ column. Elution of this solid and _SiO2 with hexane-EtOAc, 9: 1 and concentration of the eluent gave 3- {2 (S) -azido-1-oxo-2- {K ^(fe hylmethoxy) carbonyl} cyclopentyl} - ethyl [4- (S) - (1-methyl-ethyl) -2-oxazolidinone (66 g, 86%).

A solution of the above compound (13.42 g, 32.4 mmol) in THF / H ₂ O (3: 1, 608 mL) was cooled to 0 ° C. To it was added hydrogen peroxide / H ₂ O (3: 7, 16.3 mL, 518 mmol hydrogen peroxide) followed by LiOH.H ₂ O (2.36 g, 68.2 mmol). The mixture was stirred for 45 minutes at 0 ° C then quenched (quenched) ¹ 10% (wt./vol) aqueous sodium sulfite solution (400 mL). After addition of NaHCO 3 (1.93 g), the mixture was concentrated under reduced pressure. The chiral excipient was recovered (regenerated) by continuous extraction for 20 hours (aqueous NaHCO 3 / chloroform). Then, the aqueous phase was cooled to 0 ° C, acidified by the addition of concentrated HCl and then extracted with EtOAc. The extract was washed with brine, dried (MgSO 4) and concentrated under reduced pressure to give the title compound as a white solid (8.2 g, 84%). ¹ H NMR (CDCl 3) of the compound showed: δ 1.6-1.8 (m, 5H),

1.95-2.05 (m, 2H), 2.20-2.30 (m, 1H), 4.55 (s, 1H), 5.12 (s, 2H) and 7.4 (m, 5H).

The compound is used in section (c) of this example. (b) NH ₂ - (S) -CH {CH ₂ C (CH ₃ ) ₃ } CH ₂ OBzl: · H-yMeLeu-OH was reduced with LiBH ₄ / Me ₃ SiCl according to the method of A. Giannis and K. Sandhoff, Angew. Chem. Int. Ed. Engl. 28, 218, 1989, to form the amino alcohol _NH2 - (S) -CH _{CH2 C (CH3) _{3} CH2} OH. A mixture of the compound so formed (812 mg, 6.51 mmol) and

-20di-tert-butyl dicarbonate (1.42 g, 6.51 mmol) in dried THF (15 mL) was stirred for 15 minutes under nitrogen at 4 ° C and then for 4 hours at room temperature. THF was evaporated under reduced pressure and the resulting residue was dissolved in EtOAc. The solution was washed with 10% aqueous citric acid, 5% aqueous NaHCO _3, and brine. The organic portion was dried (MgSO 4 and concentrated to dryness under reduced pressure. The residue was purified by flash chromatography (SiO ₂ , eluent: hexane-EtOAc, 2: 1) to give Boc-NH-tSJ-CH 2 Cl 2 / CH 2 Cl 2 OH ( 1.23 g, 86%).

Tetrabutylammonium bisulfate (106 mg) and 50% aqueous NaOH (3 mL) were sequentially added to a solution of Boc-NH- (S) -CH {CH ₂ C (CH 3) ₃ } CH ₂ OH (1.23 g, 5.35) mmol) in benzyl chloride (13 mL). The resulting mixture was stirred for 50 minutes at 35-40 ° C, diluted with EtOAc-α-washed with 4d ₂ O-α-brine: * The organic portion was dried (MgSO ₄ ) and the volatiles were removed under reduced pressure . The resulting residue was dissolved in hexane and the solution was poured onto a SiO ₂ column. The column was eluted with hexane to remove benzyl chloride and then hexane-EtOAc (2: 1) to form Boc-NH-15-CH 2 CH 2 CH 2 CH 2 Cl 2. ¹ H NMR (CDCl 3): δ 0.95 (s, 9H), 1.42 (s, 9H), 1.30-1.55 (m, 2H), 3.42 (d, J = 4) Hz, 2H), 3.88 (broad, 1H), 4.54 (m, 3H), 7.23-7.4 (m, 5H) This compound (1.28 g, 3.99 mmol) was Dissolved in 6 N HCl / dioxane (10 mL) and stirred at 4 ° C under nitrogen for 45 min. Evaporation of the solvent gave the hydrochloride salt of the title compound (1.05 g). (c) The title compound of this example: By carrying out the coupling procedure of Example 1 and using the hydrochloride salt of NH ₂ - (S) -CH {CH ₂ C (CH ₃ ) -} CH ₂ OBzl from the previous section first of the reactant and (S) -? - azido-1 - ((phenylmethoxy) carbonyl} cyclopentaneacetic acid from section (a) of this example as the second reactant gave N - ((S) -1-benzyloxymethyl-3,3) -dimethylbutyl} - (S) -α-azido-1 - {(phenylmethoxy) carbonyl} cyclopenta Reduction of this compound with stannous chloride in MeOH by the method of N. Maiti et al., described in Tetrahedron Letters 27,1423,1986, afforded the title compound.

-21 of this example. ¹ H NMR (CDCl 3) of this compound showed; δ 0.98 (s, 9H), 1.22-2.25 (m, 12H), 3.4 (d, J = 4Hz, 2H), 3.64 (s, 1H), 4.18 ( broad m, 1H), 4.52 (s, 2H), 5.12 (s, 2H), 7.18 (d, J = 7 Hz, 1H), 7.22-7.38 broad m, 10H) .

Example 3

Preparation of (PhCH ₂ ) CHC (O) - (N-Me) - Val - Tbg-Asp (pyrroltdino) - Asp (cyPn) -MeLeucino (Formula 1, A = 2- (phenylmethyl) -3-phenylpropionyl, δ = N-Me-Val, D = Tbg, R ¹ - pyrrolidine, R ² and R ³ together with the carbon atom to which they are attached form cyclopentyl and E = NHCH ² R ⁹ ) - Z, where R ⁹ is

2,2-dimethylpropyl and Z = CH ₂ OH).

(a) Preparation of intermediate Boc- (N-Me) -Val-Tbg-Asp (pyrrolidino) -Asp (cyPn) (Bzl) -NH- (S) -CH {CH ₂ C (CH ₃ ) ₃ } CH ₂ OBzl : By making a copulation. of Example 1, the title compound of Example 2 (the first reagent) was combined with the Boc-Asp (pyrrolidino) -OH (second reagent) compound described by PL Beaulieu et al. in European Patent Application No. 461,546, published 18/12/1991, to form Boc-Asp (pyrrolidino) -Asp (cyPn) -NH- (S) -CH {CH ₂ C (CH ₃ ) ₃ } CH ₂ OBzl. This compound was immediately deprotected (6 N HCl / dioxane, -45 minutes, 4 ^U C) and under similar conditions associated with Boc-Tbg-OH to give Boc-Tbg-Asp (pyrrolidino) -Asp (cyPn) -NH - (S) -CH 2 -CH 2 CH 2 CH-jOBzl. Removal of the Boc protecting group (6 N HCl / dioxane, 45 min, 4 ° C) gave the corresponding free N-terminal amine derivative as the corresponding hydrochloride addition salt. Then, the amine derivative was coupled to Boc- (N-Me) -Val-OH under similar conditions to form. of the desired intermediate.

(b) Preparing the title compound; The product of section (a) above was converted to the corresponding free N-terminal amine derivative in 6 N HCl / dioxane (4 ° C, 45 min). The amine derivative hydrochloride addition salt (447 mg, 0.490 mmol) was dissolved in CH ₂ Cl ₂ (1.5 mL). To the solution were below

N-Methylmorpholine (200 μL, 1.83 mmol) and dibenzyl acetyl chloride (269 mg, 1.04 mmol) were added under nitrogen atmosphere. The mixture was stirred at room temperature for 7 hours and then the volatiles were evaporated under reduced pressure. The resulting residue was dissolved in EtOAc. The solution was washed sequentially with 1 N aqueous HCl, saturated aqueous NaHCO _3, and brine, dried (MgSO 4 and concentrated under reduced pressure. The resulting residue was dissolved in EtOH (5 mL) and the solution was in the presence of 10% (w / v). The catalyst was then collected on a filter and the filtrate was concentrated to dryness, and the residue was purified by reverse phase HPLC on a C- column. 18 using a gradient of acetonitrile and water, each containing 0.06% trifluoroacetic acid, to afford the title compound as * white-solid- ^{H 2} O 2 -mg); ¹ H NMR (DMSO- ^d 6); d ₆ T400 MHz MHz, while this ^J compound exists in DMSO as a 4: 1 mixture of roamers) δ 0.16 (d, J = 6.5 Hz,

0.6H), 0.22 J (6.5Hz, 2..4H), 0.71. (d, J = 6.5Hz, 2.4H). 75 (d, J = 6.5 Hz, 0.6 H), 0.80 (s, 1.8H), 0.82 (s, 7.2H), 0.87 (s, 9H) / 1.40-1.55 (m, 7H) .1 .65-2.00 (m, 8H), 2.52-3.90 (m, 13H), 2.70 (s, 2. 4H, 2.89 (s, 0.6H), 4.21 (d, J = 9.5Hz) 0.8H), 4.34 (d, J = 9.5Hz, 0.2H), 4.43 (d, J =

Hz, less represented rotamer signal, 1H), 4.65-4.73 (m, 1H), 4.82 (d, J = 10 Hz, less represented rotamer signal, 1H), 7.04- 7.40 (m, 10.8H), 7.57 (d, J = 8.5 Hz, 1H), 8.01 (d, J = 10 Hz, less rotamer overlap, 1H), 25 (d, J = 9.5Hz, 0.2H), 8.30 (d, J = 7.5Hz, 0.8H), 8.51 (d, J = 7.5Hz, 0.2H) ); FAB mass spectrum m / z: 939 (M + Na) ⁺ .

Example 4

Preparation of some exemplary intermediates for elaboration of the C-terminus of the peptides of Formula 1.

(a) NH ₂ - (R) -CH (C ₂ H 4 C (CH 3) ₃ ): To a cooled solution (0 ° C) containing

-234,4-timethyl-3-pentanone (106 g, 0.923 mol), triethylamine (513 mL, 3.68 mol) and (R) -α-methyibenzylamine (114 g, 0.940 mol) in dried benzene (1 L) a solution of TiCl ₄ (50.5 mL, 0.459 mol) in benzene (200 mL) was added at a rate such that the temperature of the mixture did not exceed 10 ° C. The mixture was then mechanically stirred for 3 hours at 40 ° C, cooled to room temperature and filtered through diatomaceous earth. This was then washed with EtOAc. The filtrate combined with the wash solution was concentrated. The resulting residue was dissolved in dried MeOH (2 L). The solution was cooled to 0 ° C and NaBH ₄ (20 g, 0.53 mol) was added portionwise while keeping the temperature of the mixture below 5 ° C. After addition of approximately 2 mL of 10% aqueous HCl, the MeOH was evaporated. The residue was dissolved in Et> 0 and the solution was washed with brine, dried (MgSO 4 and evaporated to dryness to give a reddish oil (18: 1 mixture of diastereoisomers according to NMR data) .The oil was purified by flash chromatography (SiO ₂ , eluent EtOAc) (hexane, 7:93) to give N- (1 (R) -phenylethyl) -1 (R) -ethyl-2,2-dimethylpropylamine as a liquid (110 g, 54%) which was dissolved in hexane ( To the solution was added 6N HCl in dioxane (90 mL) over 15 minutes, the resulting white solid was collected on a filter and washed with hexane to give N- (1 (R) -phenylethyl) -1 (R). ) -ethyl-2,2-dimethylpropyl hydrochloride (125 g, 97%) ¹ H NMR (CDCl 3): δ 0.55 (t _of J = 7.5 Hz, 3H), 1.14 (s, 9H), 1.54-1.95 ( m,

2H), 2.23 (d, J = 6.5Hz, 3H), 2.36-2.44 (m, IH), 4.31-4.49 (m, IH), 7.30-7.48 (m, 3H), 7.74-7.79 (m, 2H) .

A solution of this compound (41.5 g) in MeOH (120 mL) was added. mixed with 10% (w / w) Pd / C (4.0 g) and the mixture was shaken under 50 psi hydrogen pressure on a Parr hydrogenator for 48 hours at room temperature. The mixture was then filtered and the filtrate was concentrated to give the desired compound NH ₂ - (R) -CH (C ₂ H ₅ ) C- (CH ₃ ) ₃ as its hydrochloride acid addition salt as a white solid (25%). g, 100%). ¹ H NMR (CDCl ₃ ): δ 1.10 (s, 9H), 1.22 (t, J = 7Hz, 2H), 1.58-1.90 (m, 2H), 2.70-2 85 (m, 1H); 8.10-8.40 (broad s, 3H).

In the same way, however, in the procedure described above, 4,4-dimethyl-24-3-pentanone was replaced with 3,3-dimethyl-2-butanone, NH ₂ - (R) -CH (CH ₃ , C (CH _{3) 3} ) was obtained. ) ₃ .HCI.

Example 5

Inhibition of Herpes simplex (HSV-1) ribonucleotide reductase (a) Preparation of enzyme

HSV-1 ribonucleotide reductase (partially purified) was obtained from BHK-21 / C13 quiescent cells infected with HSV-1 strain F at 10 bird-forming units / cell as described by E. A. Cohen, co-authored by YJ.Geri. A., 66, 733, 1985.

(b) Determination

The assay described by P. Gaudreau et al. in J. Biol. Chem. 262, 12413, 1987, is used to determine the ability of compounds of this invention inhibit the activity of HSV-1 ribonucleotide reductase, results are expressed as the concentration of compound that causes 50% of maximal inhibition (IC _S0) of enzyme activity. The number of enzyme preparation units used in each assay was constant and based on the specific activity of the enzyme preparation. The results are relative to the activity observed in the control experiments in the absence of test substance and represent the average of four determinations that differed by less than 10% from each other.

When the compound (PhCH ₂ ) ₂ CHC (O) - (N-Me) - Val - Tbg - Asp (pyrrolidino) - Asp (cyPn) -γ MeLeucinol of Example 3 was tested according to the above assay, the concentration of this compound was found. , causing a 50% reduction in HSV-1 ribonucleotide reductase activity, ie IC ₅₀ , equal to 0.17 μΜ.

-25Example 6

Inhibition of Herpes Simplex Virus (HSV-2) replication in cell culture

Determination:

BHK-21 / C13 cells (ATCC CCL 10) were incubated for two days in 150 cm ² T-flasks (1.5 χ 10 ⁶ cells / flask) in α-MEM medium (Gibco, Canada Inc., Burlington, Ontario, Canada) supplemented with 8% (v / v) fetal bovine serum (FBS, Gibco Canada Inc). Cells were trypsinized and then transferred to fresh medium in 24-well plates to obtain 2.5 χ 10 ⁵ cells in 750 μ | media per well. The cells were incubated for 6 hours at 37 ° C to allow them to adhere to the plate. They were then washed once with 500 μΙ of α-MEM supplemented with 0.5% (v / v) FBS and incubated with 750 μ | of the same medium (low in serum) for 3 days. After this serum starvation period, the low serum medium was removed and the cells were incubated in 500 μΙ BBMT for 2-3 hours. [BBMT has been described by P. Brazeau and co-authors in Proc. nati. Acad. Sci. USA 79, 7909 (1982)]. The cells were then infected with HSV-2 (multiplicity of infection = 0.02 PFU / cell, PFU = plaque forming units) in 100 μΙ of BBMT medium. (Note: HSV-2 used was HG-52 strain, see Y. Langelter and G. Buttin, J. Gen. Virol. 57, 21, 1981; virus was stored at ~ 80 ° C). After 1 hour of adsorption of the virus at 37 ° C, the medium was removed and the cells were washed with BBMT (3 x 250 μΙ). Cells in each well were incubated in the presence of the appropriate concentration of test reagent dissolved in 200 μΙ of BBMT medium or in the absence of reagent (control). After 29 hours incubation at 37 ° C, infected cells were harvested by freezing the 1 plate at -80 ° C and then thawing. The cells in each well were from its <. surface released by the effect of melting ice fragments. After complete thawing, the cell suspensions were pooled and each well was washed with 150 μΙ of BBMT medium. The viral / sample (suspension and washing fluid) was slightly disintegrated by ultrasound

[. (sonicated) for 4 minutes at 4 ° C. Cell debris was removed by centrifugation (1000 g for 10 minutes at 4 ° C). The supernatant was pooled and stored at -80 ° C until assayed. ý; -26 virus titer.

Viral encryption was performed by a modified method of colorimetric assay of M. Langlois et al., Journal of Biological Standardization 14, 201, 1986, described in detail by R. Déziel and Y. Guindon, supra.

Thus, percent inhibition of viral outgrowths can be determined at various concentrations of test agent and the concentration of test agent (ie, peptide of Formula 1) causing 50% inhibition of viral replication, ie, EC ₅₀ , can be calculated.

When she was. the compound &quot; PhGH4 ^ HC (O) ^ (N ^ VIe) & quot ^{; was} tested in cell culture as described above ^; Val7Tb ^ rVsp (pytolidino) VAsp (cyPn) -γ MeLeucino! from Example 3, the concentration of this compound was found to cause 50% inhibition of HSV-1 replication, i.e. EC _{50 of} 3 µΜ. .

Other specific examples of compounds of Formula 1 for use in the present invention are:

(PhCH ₂ ) _from CHC (O) -N-Me) -Val-Tbg-Asp (pyrrolidino) -Asp (cyPn) -y MeLeu-OH, (PhCH ₂ ) ₂ CHC (O) - (N-Me) - Val-Tbg-Asp (pyrrolidin) -Asp (cyPn) -NHCH ₂ C (.CH _{3) 3,} and (PhCH _{2) 2} CHC (O) - (N - Me) Val-Tbg-Asp (pyrrolo [pyrrolidin ) -Asp (cyPn) -NH- (R) -CH (C ₂ H ₅ ) C (CH ₃ ) ₃ . '

Claims (20)

PATENT CLAIMS 1. A peptide of formula 1 ABD-NHCH (CH ₂ C (O) R ¹ ) C (O) -NHCH (CR ² (R ³ ) C (O) 0 H) C (O) -E where A is a 2-methyl-1-oxopropyl radical disubstituted by phenyl or monosubstituted phenyl, the monosubstituted of which is selected from the group consisting of lower alkyl, halo radical, hydroxyl and lower alkoxy; B is N (CH 3) CHR ⁴ C (O) wherein R ⁴ is lower alkyl; D is NH-CHR ⁵ C (O) wherein R ⁵ is lower alkyl or lower alkyl substituted by one carboxyl, hydroxyl, mercapto or benzyloxy group; R ¹ is lower alkyl, lower cycloalkyl, {1- (lower alkyl) - (lower cycloalkyl)}, or NR ⁶ R ⁷ wherein R ⁶ is hydrogen or lower alkyl and R ⁷ is lower alkyl, or R ⁶ and R ⁷ together with the nitrogen atom to which they are attached form a pyrrolidine, piperidine, morpholine or 4-methylpiperazine group; R ² is hydrogen or lower alkyl and R ³ is lower alkyl, or R ² and R ³ together with the carbon atom to which they are attached form a lower cycloalkyl; and E is NHR ® wherein R ⁶ is C 4-9 alkyl; lower cycloalkyl; lower cycloalkyl, substituted one or two times lower alkyl or (lower alkyl) - (lower cycloalkyl); or E is NHCH 2 R 2 -Z, wherein R ⁹ is C ^1-9 alkyl, lower cycloalkyl or (lower cycloalkyl) - (lower alkyl) and Z is CH ₂ OH, C (O) OH, C (O) NH ₂ or C (O) OR ¹⁰ wherein R ¹⁰ is lower alkyl; or a therapeutically acceptable salt thereof; wherein the term lower alkyl, either alone or in combination with another radical, denotes a straight chain alkyl radical of 1 to 6 carbon atoms or a branched chain alkyl radical of 3 to 6 carbon atoms; the term lower cycloalkyl, either alone or in combination with another radical, refers to a saturated cyclic hydrocarbon radical containing three to six carbon atoms; the term lower alkoxy means a straight chain alkoxy radical containing one to four carbon atoms and a branched chain alkoxy radical containing -28 three to four carbon atoms; the term 1- (lower alkyl) - (lower cycloalkyl) refers to a lower cycloalkyl radical bearing a lower alkyl substituent at the 1-position. A peptide according to claim 1, wherein A is A 2-methyl-1-oxopropyl radical, double substituted with phenyl, 4- (lower alkyl) phenyl, 4-halophenyl or 4- (lower alkoxy) phenyl; B is (N-Me) Val, (N-Me) 11e or (N-Me) Tbg; D is an amino acid residue of (S) -2-amino-3-hydroxy-3-methylbutyric acid or (R) -2-amino-3-mercapto-3-methylbutyric acid, or an amino acid residue selected from Val, Ile and Tbg; R ¹ is lower alkyl, lower cycloalkyl, {T- (lower alkyl) - (lower cycloalkyl)}, Ν, Ν-dimethylamino, Ν, Ν-diethylamino, pyrrolidine or morpholine *; -definici-uvedenév nároku'í; * ~ Ae "NHR ⁸⁾ R 'wherein R ⁸ is C4.3 alkyl; lower cycloalkyl, lower cycloalkyl, mono- or di-substituted by lower alkyl; or a (lower alkyl) - (lower cycloalkyl) substituent; or E is NHCH (R ®) -Z, wherein R ⁹ is C 1-4 alkyl or (lower cycloalkyl) methyl and Z is as defined in claim 1; or a therapeutically acceptable salt thereof. 3. The peptide of claim 2, wherein A is 2- (phenylmethyl) -3-phenylpropionyl, 2 - {(4-fluorophenyl) methyl} -3- (4-fluorophenyl) propionyl, 2 - {(4-methoxyphenyl) methyl} -3-phenylpropionyl; or 2 - {(4-methoxyphenyl) methyl} -3- (4-methoxyphenyl) propionyl; B is (N-Me) -Val or (N-Me) -lle; D is Val, Ile or Tbg; R ¹ is 1-methylethyl, 1,1-dimethylethyl, 1,1-dimethylpropyl, cyclobutyl, cyclopentyl, cyclohexyl, 1-methylcyclopentyl; Ν, Ν-dimethylamino, Ν, Ν-diethylamino, pyrrolidine or morpholino; R ² is hydrogen and R ³ is methyl, ethyl, 1-methylethyl, 1,1-dimethylethyl or propyl, and the carbon atom bearing R ² and R ³ is in the (R) configuration, or R ² and R ^{3 are} each independently methyl or ethyl, or R ² and R ³ together with the carbon atom to which they are attached form cyclobutyl, cyclopentyl or cyclohexyl; and E is NHR ³ , wherein R ® is 2,2-dimethylpropyl, 1 (R), 2,2-trimethylpropyl, 1 (R) -ethyl-2,2-dimethylpropyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl, 1 (R), 2,2-trimethylbutyl, 1 (R), 3,3-trimethyibutyl, -291 (R) -ethyl-3,3-dimethyl, or cyclohexylmethyl; or E is NHCH 2 R 2 -Z, wherein the carbon atom bearing R ⁹ is in the (S) configuration, R ⁹ is 1,1-dimethylethyl, 1-methylpropyl, 2-methylpropyl, 2,2-dimethylpropyl or cyclohexylmethyl and Z is CH ₂ OH, C (O) OH, C (O) NH ₂ or C (O) OR ¹⁰ , wherein R ¹⁰ is methyl, ethyl, or propyl; or a therapeutically acceptable salt thereof. 4. The peptide of claim 3 wherein A is 2- (phenylmethyl) -3-phenylpropionyl; B is (N-Me) m / a; D is Tbg; R ¹ is 1-methylethyl, 1,1-dimethylethyl, 1-methylpropyl, 1,1-dimethylpropyl, 2,2-dimethylpropyl, cyclobutyl, cyclopentyl, cyclohexyl, 1-methylcyclopentyl, pyrrolidine or morpholine; R ³ is hydrogen and R ³ is methyl, ethyl, 1-methylethyl or propyl, and the carbon atom bearing R ³ and R ³ | is in the (R) configuration, or R ² and R ³ are each independently methyl or ethyl, or R ² and R ³ together with the carbon atom to which they are attached form cyclobutyl, cyclopentyl or cyclohexyl; and E is NHR ³ , wherein R ⁸ is 2,2 ^: dimethylpropyl, 1 (R), 2,2-trimethylpropyl, 1 (R) -ethyl-2,2-dimethylpropyl, 2,2-dimethylbutyl or 1 (R) -ethyl-3,3-dimethylbutyl, or E is NHCH 2 R 4 -Z, wherein the carbon atom bearing R ⁹ is in the (S) configuration, R ⁹ is 2,2-dimethylpropyl and Z is CH ₂ OH, C (O) OH, C (O) NH ₂ or C (O) OR ¹⁰ , wherein R ¹⁰ is methyl, ethyl, or propyl; or a therapeutically acceptable salt thereof. A peptide according to claim 1, characterized in that it is selected from the group consisting of; (PhCH ₂ ) ₂ CHC (O) -N-Me) -Val-Tbg-Asp (pyrrolidino) -Asp (cyPn) -7 MeLeucinol, (PhCH ₂ ) ₂ CHC (O) -N-Me) -Val-Tbg -Asp (pyrrolidino) -Asp (cyPn) -MeLeu-OH, (PhCH ₂ ) ₂ CHC (O) - (N-Me) -Val-Tbg-Asp (pyrrolidino) -Asp (cyPn) -NHCH ₂ C (CH ₃ ) ₃ , a (PhCH ₂ ) ₂ CHC (O) - (N-Me) -Val-Tbg-Asp (pyrrolidino) -Asp (cyPn) -NH- (R) -CH (C ₂ H ₅ ) C ( CH ₃ ) ₃ . <TJ O 1 T) r " O and >: ZP ί n. 1; tn 2 C- 1 ¹ Xt X σ O ‘Ť? > czx at_ in.. and _ο b 0 YEAH O fC t and '' Ί ΓΤΛ J 1 cn 30 1 A pharmaceutical composition comprising an antiviral effective (relative to herpes virus) amount of the peptides of formula 1, or a therapeutically acceptable salt thereof, and a pharmaceutically or veterinarily acceptable carrier, 7. A pharmaceutical composition according to claim 6 wherein the peptide is selected from the group consisting of: (PhOH 2 CHCfO) -N-Me-Va-Tbg-Asp (pyrrolidino) -Asp (cyPn) -y MeLeucinol, (PhCH ₂ ) ₂ CHC (O) -N-Me) -Val-Tbg-Asp (pyrro (idino) -Asp (cyPn) -y MeLeu-OH, (PhCH ₂ ) ₂ CHC (O) - (N-Me) -Val-Tbg-Asp (pyrrolidino) -Asp (cyPn) -NHCH ₂ C (CH ₃₎ ) _3, and (PhCH _{2) 2} CHC (O) - (N - Me) -Val-Tbg-Asp (pyrrolidino) -Asp (cyPn) -NH- (R) -CH (C _{2 s)} C (CH ^. 8. A cosmetic composition comprising the peptide of claim 1, or a therapeutically acceptable salt thereof, and a physiologically acceptable carrier suitable for topical application. Use of the peptides according to any one of claims 1 to 4, or a therapeutically acceptable salt thereof, for the manufacture of pharmaceutical compositions for the treatment of herpes viral infections in mammals. Use according to claim 9, characterized in that the peptide is selected from the group consisting of: .... (PhCH ₂ ) ₂ CHC (O) - N-Me) -Val-Tbg-Asp (pyrrolidino) -Asp (cyPn) -y MeLeucinol, (PhCH ₂ ) ₂ CHC (O) - Ν-β) -ν3ΐ-Τός -Al5ρ (ργπ · οΙίΰίηο) -Α3ρ (ογΡηΗ MeLeu-OH, (PhCH ₂ ) ₂ CHC (O) - (N-Me) -Val-Tbg-Asp (pyrrolidino) -Asp (cyPn) -NHCH ₂ C (CH 3) ) ₃ , a -31 (PhCH CHCCl ^) - (N - Me) -Val-Tbg-Asp (pyrrolo [pyrrolidinylmethyl) -Asp (cyPn) -NH- (R) -CH (C _{2 S)} C (CH ^. Use of the peptides of any one of claims 1 to 4, or a therapeutically acceptable salt thereof, for the manufacture of a pharmaceutical composition for inhibiting herpes virus replication. Use according to claim 11, characterized in that the peptide is selected from the group consisting of: (PhCH3 CHCl3) -N-Me-Va-Tbg-Asp (pyrrolidino) -Asp (cyPn) -y MeLeucinol, (PhCH3) ₂ CHC (O) -N-Me) -Val-Tbg-Asp (pyrrolidino) -Asp (cyPn) -yl MeLeu-OH (Ph-CN-CHCíOJ IVleJ-Val-Tbg-AspÍpyrrolidinoJ AspícyPnJ-NHC-UP ^ ^, and (PhCH _{2) 2} CHC (O) - (N - Me) -Val-Tbg-Asp (pyrrolidino) -Asp (cyPn) -NH- (R) -CH (C ₂ H ₅ ) C (CH ₃ ) ₃ . 13. A pharmaceutical composition comprising a pharmaceutically or veterinarily acceptable carrier and an effective amount of a combination of an antiviral nucleoside analogue, or a therapeutically acceptable salt thereof, and a peptide of claim 1, comprising a ribonucleotide reductase, or a therapeutically acceptable salt thereof. 14. A pharmaceutical composition according to claim 13 wherein the nucleoside analog is a compound of formula 2. CH ₂ OCH ₂ CH ₂ OH Where R ¹¹ is a hydrogen atom or a hydroxy or amino group, or a therapeutically acceptable salt thereof. The pharmaceutical composition of claim 13, wherein the antiviral nucleoside analog is selected from the group of vidarabine, idoxuridine, trifluridine, ganciclovir, edoxudine, brovavir, fiacitabine, penciclovir, famciciovir and rociclovir. Pharmaceutical composition according to any one of claims 6, 7, 13, 14 and 15, characterized in that the composition is in the form of a preparation for topical application. . ____________________ _{_} _________________ Use of a combination of an antiviral nucleoside analogue, or a therapeutically acceptable salt thereof, and a peptide of formula 1 according to claim 1, inhibiting ribonucleotide reductase, or a therapeutically acceptable salt thereof, for the manufacture of pharmaceutical compositions for the treatment of herpes viral infections in mammals. Use according to claim 17, characterized in that the antiviral nucleoside analogue is selected from the group consisting of acyclovir, 6-deoxyacyclovir, 2,6-diamino-9 - {(2-hydroxyethoxy) methyl} purine, vidarabine, idoxuridine, tripluridine, gangciclovir, edoxudine, brovavir, fiacitabine, pencíclovir, famciclovir and rociclovir. Use of (PhCH 2 CHCl 3) - N-Me) - Val-Tbg-Asp (pyrrolidino) -Ap (cyPn) -yMeLeucinol for the manufacture of a medicament for the treatment of infections caused in mammals by Herpes simplex virus type 1 or type 2. 20. A method for producing a peptide according to claim 1, or a therapeutically acceptable salt thereof, comprising: (a) multistep linking, in sequence according to the sequence of the peptide, amino acid or derived amino acid residues, and non-peptide fragments of the peptide, wherein (33i) the side chain reactive groups or fragments are protected with appropriate protecting groups to prevent chemical reactions from occurring at this site until the protecting group is finally removed after the multi-step coupling is complete; The α-amino group of the linking reactant is protected by the α-amino protecting group, while the free carboxyl group of the reactant is linked to the free α-amino group of the second reactant; the .alpha.-amino protecting group is one of the groups that can be easily removed to allow the subsequent coupling step to take place on the .alpha.-amino group; and iii) The C-terminal carboxyl group of the amino acid residue or peptide fragment to form the C-terminal function of the protected peptide, if present, is protected with a suitable protecting group to prevent a chemical reaction from occurring at that location until the desired peptide amino acid sequence; and (b) upon completion of the coupling, elimination of any protecting groups and, if desired, performing standard conversions to obtain the peptide of Formula 1; and, if desired, converting the peptide into a therapeutically acceptable salt.